The present invention concerns a novel antibody that may be used in immunoassays of components reflecting body fluid levels of 5-hydroxytryptophol [5-HTOL=2-(5-hydroxy-3-indolyl)ethyl alcohol]. Free 5-HTOL and glucuronide and sulphate conjugates thereof are known markers for recent alcohol intake in humans. Other aspects of the invention are as indicated in the title.
5-hydroxytryptophol (5-HTOL) and 5-hydroxyindole-3-acetic acid (5-HIAA) are human metabolites of serotonin (5-hydroxytryptamine, 5-HT) via the common intermediate 5-hydroxyindole-3-acetaldehyde (5-HIAL). 5-HTOL is excreted in the urine where it mainly occurs conjugated with a glucuronic acid (T-5-G (or GHTOL), 75-95%) and, to a lesser extent, in free form or conjugated as a sulphate. 5-HIAA is also excreted in the urine. After alcohol consumption, the 5-HTOL level in various body fluids will raise from normal values that for urine are in the range of 40-650 nmol/L. The 5-HTOL/5-HIAA ratio will simultaneously become elevated from normal values that are  less than 0,01 (or  less than 10 if expressed as picomolar/nanomolar). The increase of both the 5-HTOL level and the 5-HTOL/5-HIAA ratio persist for several hours after alcohol has disappeared from the body. These findings have made 5-HTOL and the 5-HTOL/5-HIAA ratio markers of recent alcohol consumption in the treatment of alcohol dependence and in forensic medicine, e.g. during post-mortem body examination. The preference has been for measuring the ratio because 5-HTOL levels, but not the ratio, are increased also after ingestion of food rich in serotonin, and because the range for normal and abnormal 5-HTOL levels, but not the ratios, are overlapping. As an alternative also the ratio between urinary 5-HTOL and other urinary excretion products, such as creatinine, have been used.
The term xe2x80x9c15-HTOLxe2x80x9d includes 5-HTOL compounds, such as free 5-HTOL, its glucuronide (tryptophol-5-gucuronide, T-5-G) and its sulphate, if not otherwise specified.
Increased levels of 5-HTOL compounds after alcohol consumption have so far been detected in urine, plasma, cerebrospinal fluid (CSF). It is most likely that one can detect 5-HTOL compounds also in serum, tear fluid, sweat, saliva etc. The normal level of 5-HTOL compounds and the balance between free 5-HTOL and its conjugated forms may be different between various types of sample samples.
Compared to 5-HIAA, 5-HTOL is complicated to measure. It occurs in lower concentrations than 5-HIAA and the conjugated forms are in excess with a strong predominance for the glucuronide. The presently used method for measuring 5-HTOL is gas chromatography in combination with mass spectrometry (GC/MS), a method requiring expensive instruments. A high pressure liquid chromatography (HPLC) method has been developed but it suffers from the drawback that of xe2x80x9cfalse peaksxe2x80x9d, i.e. overlapping Rf for 5-HTOL and other compounds. Both methods require that the glucuronide (T-5-G) has to be converted enzymatically to free 5-HTOL before the assay. Thus there is a need for a cheap, simple and reliable method for measuring 5-HTOL compounds in various body fluids.
For earlier published articles within the field of the invention see separate list at the end of the descriptive part.
Reference may also be made to Dabadie et al, Synapse 14: 178-183 (1993), Iijima et al, Acta histochem 89: 141-156 (1990), U.S. Pat. No. 4,762,781 (Geffard) and EP-A-216162 (A/S De Danske Sukkerfabrikker)
The primary objective of the invention is to provide an immunoassay method for the measurement of a 5-HTOL compound in samples containing free 5-HTOL together with its conjugates without prior hydrolysis of endogenous 5-HTOL conjugates.
An second objective is to provide an antibody allowing for an immunoassay of a 5-HTOL compound.
A third objective is to provide derivatives of T-5-G that may be used as immunogens or as 5-HTOL analogues mimicking the structure of corresponding native 5-HTOL compound in various assays, e.g. competitive immunoassays for 5-HTOL compounds.
The Discovery Behind the Invention
The invention is based on our discovery that one can obtain antibodies that bind to a 5-HTOL compound by raising a humoral immune response in the appropriate animals using an immunogenic form of the xcex2-glucuronide of 5-HTOL and D-glucopyranosiduronic acid (i.e. an immunogenic form of 3-(2-hydroxyethyl)indole-5-O-xcex2-D-glucopyranosiduronic acid) as the immunogen.
The Inventive Antibody Preparation
The first aspect is thus an antibody specific against a 5-HTOL compound. By the term xe2x80x9cspecificxe2x80x9d is meant that the antibody during immunoassay conditions preferentially will bind to a 5-HTOL compound with no disturbing binding activity against other endogenous substances in human samples, for instance serotonin (5-HT), 5-HIAA or other structurally related substances, such as other indoles or glucuronides. In the presently preferred mode the inventive antibody preparation preferentially binds to T-5-G compared to other endogenous 5-HTOL compounds. The antibody preparation has an acceptable low binding activity to potential disturbing substances, such as other indoles and glucuronides, that may be present in body fluids. See the experimental part. Since the levels of free 5-HTOL, T-5-G and sulphated 5-HTOL all are elevated as a consequence of recent alcohol intake, useful antibodies may react with one, two or all of these 5-HTOL compounds.
The term antibody encompasses various antibody preparations having the above-mentioned specificity, such as the intact antibody and various active fragments (antigen/hapten binding fragments), such as Fab, Fabxe2x80x2, Fv, F(abxe2x80x2)2 etc. It also covers derivatized antibodies, such as antibodies to which labels have been attached covalently, such as biotin, hapten, enzymes, enzyme substrates, cofactors, fluorophors, chemiluminescers, chromophors, radioactive isotopes, metals, particles etc, and carrier molecules, such as insoluble and soluble polymers.
The inventive antibody may be a polyclonal or monoclonal preparation. It may contain a mixture of a definite number of monoclonals. It can be obtained by commonly known methods, except that our novel T-5-G derivatives (conjugates) should be used, for instance as an immunogen to raise polyclonal antibodies or as an antigen for the screening of immune response or of cell lines (e.g. hybridomas) secreting the inventive antibody or various antibody libraries from which each individual antibody component can be isolated together with the corresponding coding sequence. Thus the techniques contemplated do also encompass antibodies obtained via selection by phage display techniques. Once obtained, the inventive antibody preparation may be modified by recombinant techniques.
The best mode at the priority date for obtaining the inventive antibody preparation is given in the experimental section and utilizes as the immunogen the conjugate between keyhole limpet hemocyanine (KLH) with T-5-G as defined in patent example 3 (reaction scheme 2) and selection of appropriate hybridoma clones as described in the experimental section. The best mode inventive antibody thus preferentially binds to T-5-G among various 5-HTOL compounds present in native samples.
The Inventive 5-HTOL Analogues
The T-5-G (i.e. 3-(2-hydroxyethyl)indole-5-O-xcex2-D-glucopyranosiduronic acid) and its derivatives are novel in the sense that they never have been isolated before. Thus the broadest concept of this aspect of the invention is a 5-HTOL compound characterized in exhibiting the structure of 3-(2-hydroxyethyl)indole-5-O-xcex2-D-glucopyranosiduronic acid, optionally derivatized at one or more OH in the carbohydrate part, i.e. at an alcoholic hydroxy group (positions 2, 3 and 4) or at the carboxy group (position 6) or at the 3-hydroxyethyl hydroxyl or at the indole nitrogen.
Derivatization at an alcoholic hydroxy group may be accomplished as normally is done for glucuronide derivatives, e.g. selective alkylation or acylation at positions 2, 3 and/or 4, either before or after glucuronide formation with free 5-HTOL. Derivatization at the carboxy group of the glucuronide or of the free glucuronic acid may lead to amides and esters in which the carboxy group is further derivatized. See the experimental part.
T-5-G is preferably derivatized at the carboxy group to contain a covalently bound carrier molecule, preferably of polymeric nature, for instance an immunogenic protein carrier, such as human or bovine serum albumin, KLH or the like, or an insoluble or insolubilizable carrier polymer (support) of the type used as separation media in chromatography and immunoassays (e.g. supports made from synthetic polymers, such as polystyrene, or from polysaccharide, such as agarose, dextran, cellulose, starch and the like).
The derivatization of the T-5-G structure may also be made in order to introduce a covalently attached analytically detectable group, preferably via the carboxy group. The analytically detectable group may be of the same type as discussed for the inventive antibodies, see above.
The preferred methods of synthesizing the inventive T-5-G compounds at the priority date are apparent from the experimental part.
The Inventive Immunoassay Aspect
This aspect of the invention encompasses contacting a sample containing a 5-HTOL compound with the inventive antibody under conditions allowing formation of an immune complex comprising both a 5-HTOL compound and the inventive antibody. The conditions are then selected so that the amount of complex formed will be a measure of the amount of the 5-HTOL compound in the sample.
Determination of the complex is carried out according to methods well-known in the art, for instance either as the complex as such or as the decrease in the amount of uncomplexed inventive antibody added.
One general type of immunoassay that can be employed utilizes a labelled immune reactant that is able to form immune complexes in an amount that is related to the amount of the 5-HTOL compound present in the sample. In principle the label may be of the same general types as indicated above for 5-HTOL glucuronide analogues and antibodies.
Immunoassays utilizing labels are often divided into heterogeneous and homogeneous assays. The homogeneous variants utilize no separation step of complex-bound labelled reactant from uncomplexed labelled reactant. This makes it imperative to use a label that change its signal as a consequence of complex formation. In heterogeneous assay variants the labelled reactant that is bound in an immune complex is physically separated from the labelled reactant not bound in the complex. The heterogeneous variants thus have no demand for labels changing their signal due to complex formation, meaning that also radioactive isotope labels can be used.
Still another way of dividing immunoassays are in competitive and non-competitive (sandwich) variants. In the former case the 5-HTOL compound binding to the inventive antibody and present in the sample is allowed to compete with a T-5-G analogue for a limited amount of the inventive antibodies (the 5-HTOL compound reactive with the antibody plus the T-5-G analogue), whereafter the amount of complex between the T-5-G analogue and the inventive antibody is determined and related to the amount of the 5-HTOL compound in the sample. The T-5-G analogue is in this case preferably a labelled or a carrier bound T-5-G derivative as defined above. In case the T-5-G analogue is the labelled reactant, the inventive anti-5-HTOL antibody used may be in insolubilized form (for instance linked to a support) or soluble form. An alternative is a variant in which an insoluble T-5-G analogue is competing with a 5-HTOL compound of the sample for a limited amount of the soluble inventive anti-5-HTOL antibody.
The preferred immunoassay variants at the priority date encompass heterogeneous competitive immunoassays as described above. See also the experimental part.
Among assay variants utilizing no labelled reactant may be mentioned biosensor techniques where binding between antigen/hapten and antibodies is occurring at a surface.
The sample may be serum, plasma, urine, CSF or any other sample that may contain one or more 5-HTOL compounds.
The temperature and pH-conditions that are normal for immunoassays are applicable, i.e. 0-40xc2x0 C., with preference for 15-35xc2x0 C., and pH 4-9, with preference for pH 5-8.
The result of the inventive immunoassays may be used in diagnostic methods for determining recent alcohol consumption of an individual in the same way as done earlier for 5-HTOL compounds (see the introductory part). This means that in the preferred variants of the inventive diagnostic methods found levels of 5-HTOL compounds may be put into relation with some other compound, e.g. the levels of 5-HIAA or creatinine. For immune assays the amount of immune complex formed or any other measure of the amount of 5-HTOL compounds in the sample may of course be directly related to recent alcohol consumption equally well as the level a 5-HTOL compound as such. The diagnostic method is particularly well adapted to human individuals.